Lymnaea stagnalis  cannot be attributed to the effects of serotonin.The role of serotonin in the immediate and delayed influence of physical exercise on brain functions has been intensively studied in mammals. Recently, immediate effects of intense locomotion on the decision-making under uncertainty were reported in the Great Pond snail, tagnalis . In this The influence of physical exercise on brain function has been thoroughly investigated in various mammalian species . It has In mammals, physical activity affects neuromodulatory and neurotrophic systems , namely,Lymnaea stagnalis snail are, in many aspects, similar to those observed in rodents and humans, with a decrease in defensive responses, an increase in general activity  whether the behavioral effects of the exercise persist for 2 h after its termination; (2) what metabolic changes in the serotonergic system accompany intense locomotion and recovery; and (3) how serotonergic ligands influence the above-described behavior of Lymnaea stagnalis snails were taken from a breeding colony initially obtained from the Netherlands (1992) and mixed with specimen from wild population of the Oka River, Moscow region, Russia. They were fed daily on lettuce ad libitum and kept in dechlorinated tap water at room temperature.The Enhanced motor activity was evoked as in  by puttiWe used the same test and set up as in The movements of each snail were recorded at 15 frames per second for 15 min with the Microsoft LifeCam HD-3000 video camera. The recordings were video-tracked using the EthoVision XT software  and independently scored manually with RealTimer . The camera was placed under the transparent plastic bottom of the arena. The traces left by the crawling snails were removed with a clean paper towel before each new test.During the analysis, a centered square zone (22 × 22 cm) that limited the track and scoring analysis was defined . This zo2, and the pedal and cerebral ganglia were dissected out of the ice-cold Lymnaea physiological solution. Each paired ganglion was homogenized in 50 μl ice-cold 0.1M perchloric acid containing 5 ng of N-ω-methyl-5-hydroxytryptamine oxalate  as an internal standard. After centrifugation of the homogenate , for 30 min), 40 μl of supernatant was collected. Using high-performance liquid chromatography with electrochemical detection , we measured the following compounds in each of the four groups: (1) serotonin (5-HT); (2) a precursor of 5-HT ; and two catabolites of 5-HT, (3) N-acetylserotonin (Nac-5-HT) and (4) 5-hydroxyindole acetaldehyde (5-HIAA). The mobile phase containing 0.18M chloroacetic acid and 16 μM disodium EDTA was adjusted to pH3.6 with NaOH. Sodium-1-octanesulfonate at 1.85 mM as an ion-pair reagent and acetonitrile (CH3CN) at 8.40% (v/v) as an organic modifier were added to the mobile phase solution. The supernatants of samples were injected directly onto the HPLC column  heated to 30°C in a column oven. A glass carbon electrode  was used for electrochemical detection . The detector potential was set at 890 mV versus an Ag/AgCl reference electrode. The chromatographs were acquired using the computer program PowerChrom .5-HT and its precursor and catabolites were measured as described previously , 2018. B2, 8 mM MgCl2, 10 mM Tris, pH7.6) containing the drug (ketanserin) 15 min prior to the test. The control snails for the experiments with 5-HTP and 5-HT were placed into the 300 ml containers filled with water taken from the breeding stock for 2 h. Snails injected with 100 μl vehicle  comprised the control group in the experiment with the serotonin antagonist ketanserin. The drugs were administered in the following concentrations: 5-HTP (0.1 mM), 5-HT (0.1 mM), ketanserin (0.02 and 0.1 mÌ). All drugs were obtained from Sigma Aldrich . The test procedure was the same as in Experiment 1 (decision-making in the novel environment).Two different procedures of drug administration were used: (1) immersion of snails in groups of four (5-HT experiment) or individually (5-HTP experiment) into water (300 ml) taken from the breeding stock, with the drug added, for 2 h prior to the test, or (2) injection of 100 μl physiological solution . The values presented in the figures are given as the median with upper and lower quartiles.The significance of the differences was tested by the non-parametric Mann–Whitney Lymnaea results in the facilitation of decision-making and acceleration of locomotion when snails are placed in the novel environment  for 2 h after exercise, control snails taken directly from their breeding colony  and control snails that resided in 300 ml containers for 2 h  were used as three groups in this experiment. The third group  was used since the novelty by itself produces locomotor arousal in snails.The snails that rested in 300 ml aquatic containers filled with the water taken from the breeding stock , however, the ER snails made fewer turns prior to decision-making  = 13.01, p = 0.0015; post hoc tests ER/Ch p = 0.0013; ER/Cn p = 0.051; Ch/Cn p = 0.79) and had higher speed of locomotion compared to the control group taken from the breeding stock  = 7.5, p = 0.0234; post hoc tests ER/Ch p = 0.020; ER/Cn p = 0.2; Ch/Cn p = 0.9). Recently, we reported that locomotor arousal developed more quickly in the ER snails than in the Cn group  = 9.1, p = 0.010, post hoc tests ER/Ch p = 0.011; ER/Cn p = 0.1; Ch/Cn p = 0.9). The ratio of light/dark choice was approximately the same in all groups ca. 75/25%  to validate the delayed effect of exercise  on the performance of turns. ER snails made significantly fewer turns .We performed an additional set of experiments with only one control group kept in novel conditions .Four separate groups of snails were used : (1) conp < 0.001) than in all other groups  = 29.16, p = 0.0001, post hoc tests ER/Cn p = 0.0016; ER/E p = 0.002; ER/EE p = 0.0001; E/Cn p = 0.9; EE/Cn p = 0.79).The most prominent changes in the content of the substances being investigated were observed in the group that rested after exercise ER, . The conN = 46) = 13.08, p = 0.0045, post hoc tests ER/Cn p = 0.029; ER/E p = 0.49; ER/EE p = 0.004; E/Cn p = 0.9; EE/Cn p = 0.85).Similarly, the 5-HIAA content was the highest in the ER group, with highly significant differences compared to the control and the 4 h -exercised (EE) snails  = 12.8, p = 0.0051, post hoc tests ER/Cn p = 0.019; ER/E p = 0.04; ER/EE p = 0.01; E/Cn p = 0.9; EE/Cn p = 0.9].The Nac-5-HT content was significantly higher in the ER group compared to all other groups . In the ER group, serotonin showed the lowest concentration, but the tendency did not reach the level of significance  = 7.94, p = 0.0473, Post hoc tests indicated a significant difference between the E and ER groups, p = 0.043).The 5-HT content in the cerebral ganglia changed significantly within the four groups .The 5-HIAA content was significantly higher in all three experimental groups compared to the control  and as a tendency compared to the control .The Nac-5-HT content was higher in the ER and EE groups compared to the E group [Kruskal–Wallis test: H  demonstrated a more rapid onset of activity compared to the control group (n = 27). The delay of the first movement was significantly smaller in the experimental snails . There were no differences in the rotational behavior, which is considered to be the orienting phase of the behavior under uncertainty, preceeding the decision-making process. The mean total rotation did not differ between the two groups . The ratio of light/dark choice was similar in both groups.5-HTP is known to increase the speed of locomotion in Lymnaea . Here wen = 24 versus n = 30 in the control group). The delay in the first movement was significantly smaller . We hypothesized that there were no statistically significant differences in the mean speed (p = 0.055), because 5-HT- treated snails tended to spend more time in the orienting phase of the behavior (performed more turns). To check this possibility we measured the mean velocity during the phase of the directional crawl (6–8 min after the start of the experiment). It was indeed significantly higher than in the control group  or 0.1 mM (n = 13) ketanserin 15 min prior to the test. The control groups  received 100 μl of physiological solution. Snails, injected with 0.02 mM ketanserin demonstrated the tendency for later onset of activity . The effect was significant in the group that received 0.1 mM ketanserin . Higher concentration of ketanserin decreased total rotation , which never occurred in any other groups  were observed  but not the result, the choice between the light and dark sides of the arena. For a snail, which has a very low speed of locomotion, in the situation of limited time and high uncertainty, long exploration of the environment with frequent changes of direction is hardly the best strategy. Investing time and energy into crawling in the chosen direction will make the survival more probable. Preadaptation, as we propose, increases the level of confidence in ambiguous situations  .Lymnaea . By contrast, in the pedal ganglia, the content of serotonin did not statistically change in the ER group (and it tended to be lower than in other groups). Therefore, serotonin metabolism seems to be differently regulated in different parts of the CNS in mollusks. Presumably, the change in distribution helps to cope with different needs for serotonin by different neuronal ensembles in various behavioral contexts.The absence of differences in the content of serotonin in the pedal ganglia in the ER group compared to control was accompanied by a 10-fold increase in the 5-HTP concentration and a significant increase in the content of both catabolites of serotonin. It is likely that the increased catabolism of serotonin and the termination of its synthesis could be responsible for the effects of rest described above. Our data point to aromatic amino acid decarboxylase (AAD) activity as the main source of the observed changes. It is likely that transient inhibition of AAD in the rest-after-exercise group was responsible for the accumulation of 5-HTP. This possibility can be experimentally verified in the future.Lymnaea earlier, specifically, after food deprivation  after exercise remain to be found in Lymnaea. Therefore, the effects of exercise on the facilitation of decision-making are likely to be mediated by multitransmitter neuromodulatory control in mollusks.We can conclude that the results of our behavioral, pharmacological and biochemical experiments suggest that serotonin is likely to play a role in the effects of exercise on the The datasets generated for this study are available on request to the corresponding author.This study was carried out in invertebrate animals, the pond snails Lymnaea stagnalis, in accordance the protocol has been approved by the responsible authorities of our institutions.HA performed HPLC experiments and edited the manuscript. MM performed behavioral experiments . BB performed behavioral experiments (5-HTP and ketanserin effects). YT performed behavioral experiments and made dissections for HPLC analysis. DV performed analysis of behavioral data using the EthoVision software. IZ participated in behavioral experiments and discussed results. EI analyzed the results and edited the manuscript. VD planned the experiments and wrote the present manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The gap-prepulse inhibition of the acoustic startle reflex has been widely used as a behavioral method for tinnitus screening in animal studies. The cortical-evoked potential gap-induced inhibition has also been investigated in animals as well as in human subjects. The present study aimed to investigate the effect of age on the cortical N1-P2 complex in the gap-prepulse inhibition paradigm. Fifty-seven subjects, aged 20 to 68 years, without continuous tinnitus, were tested with two effective gap conditions (embedded gap of 50- or 20-ms duration). Retest sessions were performed within one month. A significant gap-induced inhibition of the N1-P2 complex was found in both gap durations. Age differently affected the inhibition, depending on gap duration. With a 50-ms gap, the inhibition decreased significantly with the increase in age. This age-inhibition relationship was not found when using a 20-ms gap. The results were reproducible in the retest session. Our findings suggest that the interaction between age and gap duration should be considered when applying the gap-induced inhibition of the cortical-evoked potential as an objective measure of tinnitus in human subjects. Further studies with tinnitus patients are warranted to identify gap duration that would minimize the effects of age and maximize the difference in the inhibition between those with and without tinnitus. Tinnitus, which affects 10–20% of the world’s population, involves the perception of phantom sounds in the absence of a physical stimulus . DespiteThe gap-prepulse inhibition of the acoustic startle (GPIAS) has been used as an objective way of screening tinnitus in animals –10. GPIAAlthough the pathogenesis of tinnitus is not fully understood, animal studies have shown that tinnitus is associated with increased synchrony in the dorsal cochlear nucleus, inferior colliculus, and the auditory cortex , 20, 21.Auditory temporal processing refers to the capacity to precisely detect the temporal features of sounds . Variousn = 6) and subjects who showed noisy ALR with unclear N1-P2 (n = 4) were excluded by eye inspection. A total of 57 subjects  with a mean age of 43.7 years  participated in the test session. Of the 57 subjects, 46  participated in a retest session; the mean time interval between test and retest sessions was 27 days (SD: 32.6). Details of the subject characteristics are presented in Sixty-seven adults  were recruited from August, 2015 to December, 2017 via poster ads or word of mouth in Seoul National University Hospital, Seoul, Korea. Our sample aimed to represent normal subjects of different ages and genders matched to the demographic characteristics of tinnitus patients. Structured histories were obtained to assess pre-existing medical conditions, with a focus on hearing and tinnitus. None of the subjects were found to have suffered from tinnitus or any other otological/neurological disorders. The inclusion criteria of hearing thresholds at 1 kHz and 8 kHz frequencies were ≤30 and ≤70 dB HL (hearing level), respectively. These thresholds were selected based on the output stimuli sound level and the maximum output limitation of the experimental equipment. To evaluate subjects’ eligibility, all subjects were tested by pure tone audiometry with standardized devices and methods  across six different frequencies . A commercial audiometer  was used for the pure tone audiometry. Subjects having hearing thresholds higher than the inclusion criteria , and all participants have signed a written informed consent form. This study followed the principles expressed in the 1964 Declaration of Helsinki and its later amendments.gap-intense sound stimulus and the condition with no preceding gap is referred to as the no-gap-intense sound stimulus. In the gap-intense sound stimulus, a temporal gap of 50 or 20 ms was inserted into the background sound. The inter-stimulus interval, or time between the end of the temporal gap and the onset of the intense sound, was set to 100 ms. This is considered an optimal interval to elicit sensorimotor- and sensory-gating  and an intense sound of 1 kHz with an intensity of 65 dB SL . These fy-gating , 45, 46. stimuli . An ER-2gap-intense sound stimulus and no-gap-intense sound stimulus were recorded in pseudorandom order during each measurement. A single epoch was 700 ms long with the above-mentioned components of sound stimulus and 100-ms pre-stimulus onset time, to allow for baseline corrections. During each of the two sessions (test and retest), two measurements, ~10-minute long each, were taken from each participant–one that contained the 50 ms embedded gaps and the other with the 20 ms embedded gaps. The acquired signals were filtered with an analog band-pass filter with a cutoff frequency of 1 to 100 Hz. The signals then went through analog-digital conversion (ADC) with a sampling frequency of 1 kHz. Further, a digital low-pass Butterworth filter with a cutoff frequency of 30 Hz was used for waveform smoothing. In a single measurement, epochs were ensemble-averaged to produce two auditory late response waveforms corresponding to the gap-intense sound stimuli and no-gap-intense sound stimuli. Epochs exceeding ± 50 μV, which were observed in less than 10% of all measurements, were excluded.All measurements were conducted in a soundproof booth. Participants were seated in a reclining armchair and instructed not to move, close their eyes, or fall asleep. They were also told not to pay attention to any sound stimuli (passive listening). Muted clips of historical, scientific, or medicine-based videos were played to divert the subjects’ attention away from the sound stimuli and prevent drowsiness. Highly violent, emotional, or fearful video clips were excluded. Each measurement was followed by a three-minute break; however, for drowsy or fatigued subjects, breaks longer than 3 min were allowed before acquiring the subsequent measurement. To average out the influence of the visual stimuli, visual changes were not synchronized with the sound stimuli. Before attaching electrodes to each participant, we abraded the skin to lower the skin-electrode impedances to below 5 kΩ. Electrodes were placed on the mastoid [ipsilateral (A1 or A2) to the earphone] as a reference electrode, and on the forehead (Fpz) as a ground electrode. Adhesive silver-silver chloride electrodes were used . One active 10 mm gold cup electrode  with conductive paste was placed on the vertex (Cz) as an active electrode . A totalgap-intense sound stimuli (Gap) and that of the no-gap-intense sound stimuli (No-Gap). We also calculated the Gap/No-Gap ratio, defined as the ratio between N1-P2 amplitude during Gap stimulation to that during the No-Gap stimulation. The inhibition deficit was indicated by a high Gap/No-Gap ratio. Offline data processing was performed using MATLAB . First, a two-tailed one-sample t-test was performed to determine whether a significant inhibition occurred under each condition. The hypothesized population mean was set to 1.0, meaning there was no difference between Gap and No-Gap responses. Next, we applied a linear mixed-effects model, with the Gap/No-Gap ratio as the dependent variable and the age (continuous variable) and two gap durations  as the independent variables. Pearson correlation coefficients between the age and the Gap/No-Gap ratio for each session were also calculated. For comparison purposes, correlation analysis between age and N1-P2 amplitude in the condition with no preceding gap was performed. The test-retest reliability of the Gap/No-Gap ratio and the N1-P2 amplitude were evaluated by calculating correlation coefficients between sessions. Furthermore, the relationship between age and the hearing thresholds was analyzed because the present study had wide inclusion criteria regarding hearing levels. Pearson correlation coefficients between the hearing threshold and the Gap/No-Gap ratio for each session were also calculated. An independent t-test to compare the Gap/No-Gap ratios between male and female subjects was done to analyze the effect of gender, which could be another confounding factor. Last, the trend of the peak-to-peak amplitudes of the responses over the stimulus repetitions for each participant was analyzed to determine whether adaptation occurred in the ALR measurement. Continuous variables are presented as mean ± standard error of the mean (SEM). SPSS  was used for statistical analysis. Since it was not possible to determine the expected effect size for the linear mixed model, we did not perform a power analysis. However, it should be noted that the sample size required to determine a 0.4 difference in correlation coefficients is approximately 47  . With a 20-ms gap, the Gap/No-Gap ratios in the test and retest sessions were 0.81 ± 0.04 and 0.78 ± 0.03, respectively. One-sample t-tests confirmed significant gap-induced inhibition in both sessions .F1,55 = 2.159, P = 0.147; retest: F1,44 = 2.29, P = 0.137]. However, an interaction between gap length and age was observed in both sessions . That is, Gap/No-Gap ratio significantly increased with age, but only within a certain gap length. On test data, using a range estimate for the standard deviation, the effect of age on the Gap/No-Gap ratio was not significant with a 20-ms gap . However, a significant effect of age on the Gap/No-Gap ratio was found when the gap was set to 50-ms . That is, the Gap/No-Gap ratio significantly increased with age when the gap was 50 ms, but not when it was 20 ms. An identical trend was also found in the retest data. With a 20 ms gap, the effect of age on the Gap/No-Gap ratio was not significant , but it was significant with a 50 ms gap .Although there was a trend for the 20 ms gap to produce less inhibition than the 50 ms gap, the expected main effects of gap length in the two sessions were not significant . However, no correlations were found with a 20-ms gap duration, as shown in r = 0.18, P = 0.178; retest: r = 0.13, P = 0.384].no-gap-intense sound stimulus versus age, there was a weak but significant trend for younger subjects to have larger amplitudes with a large between-subjects variance .As shown in r = 0.25, P = 0.087]. With a 20 -ms gap, however, the Gap/No-Gap ratios between test and retest sessions had significant positive correlation . A significant positive correlation was found in the N1-P2 amplitudes between test and retest sessions as shown in r = 0.69, P < 0.001].r = 0.39, P = 0.003; 8 kHz: r = 0.59, P < 0.001]. No significant correlations between the hearing threshold at 1 kHz and the Gap/No-Gap ratio with a 50 ms gap duration were found in either sessions . No significant correlations were found with a 20 ms gap duration in either session as well . Similarly, no significant correlation between the hearing threshold at 8 kHz and the Gap/No-Gap ratio were found in either sessions , as was the case with a 20 ms gap duration in either session . Furthermore, no gender effect was found. With a 50- ms gap, no significant difference were found in either session . With a 20 ms gap, similarly, no significant differences were found in either sessions . In addition, as shown in The hearing thresholds at 1 and 8 kHz had a significant positive correlations with age [1 kHz: In the present study, we hypothesized that age could be a crucial confounding factor when investigating the GPI paradigm with a cortical long-latency response as an objective measure of tinnitus. Previous human studies with the GPI paradigm did not look into this factor, focusing on other factors such as the effect of gap durations , 17, 36.Although we focused on a specific GPI paradigm, our findings related to the 50-ms gap condition are consistent with previous reports on the effect of age on general temporal processing. Temporal processing functions can be analyzed through the conventional gap detection test –54. PrioWhy does age have no effect when using a 20 ms gap? First of all, the 20 ms gap is a more difficult condition in terms of temporal processing. It seems that the 50 ms gap is an easy condition for pre-attentive auditory processing in young subjects who can comfortably detect the gap, resulting in a very strong inhibition. When the subject grows older, the 50 ms gap is not as easily or readily detected. The temporal processing power of older subjects might need to reach its limits to barely detect the gap, resulting in a small inhibition , 67. If A further study is needed to assess the influence of age on the GPI in tinnitus patients. The background sound frequency effect should also be investigated because responsiveness to a presented stimulus can be influenced by the associated background frequency . To deveWe found an interaction between age and the embedded gap duration in the GPI paradigm in association with the pre-attentive cortical-evoked response. The age of the subject had a significant effect on the Gap/No-Gap ratio when the gap was long (50 ms), but not when it was short (20 ms). The ceiling effect in task difficulty is presumed to be the reason for this difference. Furthermore, the shorter onset timing of the inhibition cue might allow for a lower top-down modulation probability. Our findings may contribute to the development of an objective test that could draw a clear distinction between tinnitus patients and healthy controls, ultimately impacting both rigorous research and clinical practice.


Vitex agnus-castus (VA-C) leaf extracts.Candida infections are becoming more drug resistant; it is necessary to search for alternative medications to treat them. Therefore, the present study estimates the anticandidal activity of Candida species . The minimum inhibitory concentration (MIC) was estimated using the two-fold dilution method and the minimum fungicidal concentration (MFC) was determined using the classic pour plate technique. The MFC/MIC ratio was calculated to estimate the microbicidal or microbiostatic activity. A gas chromatography mass spectrometer was used to screen the phytochemicals of the VA-C leaf extracts .We used the agar well diffusion method to assess the anticandidal activity of three different VA-C leaf extracts  against three Candida species and the inhibition activity depended on the solvent used and the Candida species. The results showed that C. tropicalis was the most highly inhibited by all extracts followed by C. albicans and C. ciferrii. The MIC values were 12.5–25 µg/ml, and MFC values were 25–100 µg/ml. The ratios of MFC/MIC were two-fold to four-fold which was considered candidacidal activity. Ninety-five phytochemical compounds were identified by the GC-MS assay for the VA-C leaf extracts. The total number of compounds per extract differed. Methanol had 43 compounds, ethanol had 47 compounds, and water had 52 compounds. The highest compound concentrations were: 4,5-Dichloro-1,3-dioxolan-2-one in ethanol and methanol, 1H-Indene, 2,3-dihydro-1,1,2,3,3-pentamethyl in ethanol, Isobutyl 4-hydroxybenzoate in methanol, and Benzoic acid and 4-hydroxy- in water. These phytochemical compounds belong to different bioactive chemical group such as polyphenols, fatty acids, terpenes, terpenoids, steroids, aldehydes, alcohols, and esters, and most of which have anticandidal activity.All VA-C extracts ethanol, methanol, and water were significantly inhibited the growth of the test Candida species at low concentrations. However, appropriate toxicology screening should be conducted before use.VA-C leaf extracts may be useful alternatives to anticandidal drugs, based on their effectiveness against all test  Candida infections is on the rise, which is concerning due to their virulence, ability to survive in extreme environments, and resistance to antifungal agents   . The ratiabetic) . Candidiiabetic) . Genus Ca krusei .Vitex is one of the largest of the 250 genera in the family Verbenaceae found worldwide (Vitex agnus-castus (VA-C) and its safety as a medicinal plant are well stated . C. albicans strains and they found that the ethanol extract was more effective than the aqueous extract against C. albicans strains. However, the effect of VA-C leaf extracts of against human Candida species has not been well-studied.Antifungals have a broad range of applications but it is difficult to determine the ideal treatment regime because their use can be limited and is often accompanied by side effects. The indiscriminate use of antibiotics has led to an increased resistance to these types of medications . Accordiorldwide . The thel stated . Previoul stated . Other sl stated . A few sl stated . The antl stated . The antl stated  as well l stated . These sCandida species . We determined the phytochemicals of these extracts using Gas Chromatography-Mass Spectrometry (GC-MS).Therefore, this study aims to investigate the anticandidal activity and efficiency of VA-C leaf extracts  against the three most frequently isolated Vitex agnus-castus VA-C leaves were collected from a private garden in Dammam City, Saudi Arabia belonging to Ibtisam Mohammed Ababutain. The plant was identified according to Vitex agnus-castus leaves were washed with tap water and left to dry for 2 days at room temperature in a well-ventilated room using a fan to speed up the drying process, then ground to a fine powder. Maceration method described by Candida tropicalis and Candida albicans were provided by King Fahd Hospital, Al Khobar, Kingdom of Saudi Arabia. Candida ciferrii was obtained from the Biology Department, College of Science, Imam Abdulrahman Bin Faisal University.Three different prepared VA-C leaf extracts with a 20% (mg/ml) concentration were screened for their anticandidal activity using the agar well-diffusion method  against Candida species were prepared from new cultures in potato dextrose broth (PDB). A Biomerieux DensiCHEK plus meter device was used to adjust the cell suspension turbidity at 1–2 × 106 CFU/ml, which represents 0.5 McFarland standards. Each Petri dish was inoculated individually with 0.5 ml of the previous suspension. Melted potato dextrose agar (PDA) was poured over the inoculums, and the plates were rotated to ensure even distribution of the inoculums then left to harden at room temperate for 5 min. Five wells were made on the inoculated PDA using a 6 mm sterile cork-borer. Each well was filled with 100 µL of the plant extracts. Positive and negative controls were included; nystatin (10 mcg) was used as the positive control and DMSO was used as the negative control. The plates were incubated at 37 °C for 24 h. The anticandidal activity of the plant extracts was estimated in millimeters (mm) using a ruler and measuring the free growth zones around the wells. The experiments were performed in three replicates to ensure the reliability of the results.Inoculums of the Candida inoculums at a concentration of 1–2 × 106 CFU/ml were transferred to the wells to make a final concentration of 50%. We used growth media with the Candida inoculum in well 11 and growth media with plant extracts in well 12, as positive and negative controls, respectively. The turbidity was examined by the naked eye after an overnight incubation period at 37 °C and the lowest concentration of plant extract showing no Candida species growth was recorded as MICs. All experiments were performed in three replicates.The minimum inhibitory concentration (MIC) of VA-C leaf extracts was estimated using the two-fold dilution method  as well Candida species growth from previous MIC experiments were transferred to Petri dishes, then 15 ml of melted PDA was poured over it and gently rotated and left to solidify. Inoculated Petri dishes were incubated at 37° for 48 h. The lowest concentration that showed no visible Candida species colonies were recorded as MFC  . Concentd as MFC . All expThe anticandidal efficiency of VA-C leaf extracts  was determined by calculating the ratio of MFC/MIC according to We analyzed the bioactive compounds of all three VA-C leaf extracts  with a gas chromatography-mass spectrometer  model QP2010 SE, with a 5 Sil MS 5% diphenyl/95% dimethyl polysiloxane capillary column  using the method previously described in Candida species was conducted using one-way ANOVA test. A P-value of <0.01 was considered statistically significant. Statistical data were analyzed using Statistical Packages for Software Sciences . The effects of the ethanol extract against C. tropicalis, C. albicans and C. ciferrii were significantly higher compared to water and methanol extracts at P = 0.01, P = 0.037 and P = 0.047, respectively  as a positive control against C. albicans, where the inhibitory effect for the positive control was higher than all extracts against C. tropicalis.Our results showed that alcohol extracts (methanol and ethanol) and aqueous extract have the ability to inhibit the growth of all tested Candida species were between 0.78 µg/ml and 1.56 µg/ml, which represent dilution 7 and 8, respectively. The values of the aqueous extract were between 6.25 µg/ml and 1.562 µg/ml, which represent dilutions of 5 and 7, respectively.We found that MIC showed that the ethanol extracts of VA-C were relatively higher than water and methanol. MIC values were between 12.5 µg/ml and 25 µg/ml for ethanol and represented a dilution of 4 and 3, respectively. For water and methanol the MIC values are specified at 25 µg/ml which represents dilution 3. Our results are similar to those of C. tropicalis with the value of MFC 25 µg/ml and the aqueous extract was less effective on C. albicans, with a value of 100 µg/ml.There was a convergence of MFC values, which represents only the three dilutions from 1 to 3 (100 µg/ml and 25 µg/ml), respectively. The VA-C extract of ethanol was the most influential on Candida species are generally opportunistic and affect the group of people with low immunity so antibiotics that are prescribed are generally more effective if they are of the fatal type. Therefore, the inhibitory efficiency of the VA-C extract was estimate using the ratio between MFC and MIC. Our results showed that the ratio of MFC/MIC between two-fold to four-fold have a candidacidal effect  or inhibiting its growth (microbistatic) . Antibiol effect . To our We found that the extracts of VA-C differed in their inhibitory effect according to the type of solvent and this is may be due to the difference in the degree of polarity between the solvent. Water has the highest polarity of 1,000 followed by methanol (0.762) and finally, ethanol (0.654). The compounds extracted by these highly polar solvents differ in quantity and quality . Many stThe GC-MC analysis result revealed that all three VA-C extracts were rich in chemical compounds that act as an anti-inflammatory, anticancer, anti-Alzheimer, anti-diarrheal, anti-diabetic, anti-viral, antioxidant, anti-allergic, nematicide, antibacterial, antifungal. These extracts are also used as food preservatives and flavorings, as previously found in other published works . SeveralCandida species including the cell wall, cell membrane, and mitochondria enzymes. Some of these compounds may reduce or prevent the virulence factors, including adhesins, enzymes production, germ tubes , biofilm formation, and quorum sensing (Candida cell membrane leading to permeability of the cell contents (Candida cell (Candida species (C. albicans and may prevent biofilm formation (C. albicans cell growth by affecting the membrane and preventing adhesins, biofilm formation, and germ tube formation (The inhibitory activity of VA-C extracts maybe attributed to the presence of important bioactive compounds , which m sensing . Our rescontents , inhibitida cell  and inhiida cell . Fatty a species . Terpeneormation . Terpenoormation .Candida species despite different species. Accordingly, VA-C leaf extracts may inhibit the growth of Candida species in general, compared to antifungals that affect a specific species or a strain of species and require an accurate diagnosis of the Candida isolation to choose the appropriate antifungal. The inhibitory activity of the ethanol solvent was better than methanol and water, which may indicate the importance of choosing the appropriate solvent to extract phytochemicals with high inhibiting effectiveness and in higher quantities. Moreover, our results showed that the extract had a candidacidal effect on test Candida species at low concentrations, which may reduce the side effects of the extract. VA-C leaf extracts are advantageous, and a promising component that can be used to develop an alternative anticandidal agent. Further studies are required to assess the toxicity, genotoxicity and mutagenicity of VA-C extracts and prove their safety for human use.Our results showed that VA-C leaf extract is rich in bioactive compounds with broad spectrum activity that inhibited all the tested 10.7717/peerj.10561/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.10561/supp-2Supplemental Information 2Click here for additional data file.


Epithelial ovarian cancer (EOC) is a lethal disease due to late diagnosis and lack of effective screening methods. MicroRNA (miR/miRNA) plays an important role in ovarian carcinogenesis and may serve as a non-invasive biomarker for EOC. This study aimed to assess miR-141 expression in the blood plasma of patients with EOC and healthy subjects and determine its association with the clinical stage of EOC.−ΔΔCt methods. The data were analysed using Mann-Whitney test.This cross-sectional study used blood plasma from 30 newly diagnosed untreated patients with EOC and 25 healthy subjects. The mean age was 47.73 (SD = 10.29) years for EOC and 44.48 (SD = 16.14) years for healthy subject. The total RNA was isolated from blood plasma and reversed transcribed to obtain cDNA. The expression of miR-141 was measured by real-time quantitative polymerase chain reaction (qRT-PCR), and calculated using 2P < 0.001). Expression of miR-141 in the advanced stage was upregulated 4.2 fold compared to the early stage (P < 0.001).The expression of miR-141 was upregulated 8.41 fold in the blood plasma of EOC patients compared to healthy controls (The miR-141 was upregulated in the blood plasma of EOC and associated with an advanced stage of disease, suggesting it has potential as a biomarker for EOC detection. Ovarian cancer is one of the most common and the most deadly gynecological malignancies among women around the world –3, with EOC develops from the malignant transformation of the epithelial cells of the ovarian surface, peritoneum, or fallopian tube . EOC is The International Federation of Gynecology and Obstetrics (FIGO) staging system is widely used in clinical practice to determine the clinical stage of ovarian cancer . The FIGOne of the main factors contributing to the high mortality rate is the advanced stage of the disease at the time of diagnosis . More thCurrent strategies and methods for EOC detection include the pelvic examination, transvaginal ultrasonography (TVS) and measurement of several molecular biomarkers , 20, 21.Recently, only CA-125 and human epididymis protein 4 (HE4) have been approved by the Food and Drug Administration (FDA) for monitoring treatment and detecting disease recurrence , 26, 27.MicroRNA (miR/miRNA) are small non-coding RNA molecules with a length of 19–24 nucleotides, phylogenetically conserved in various species, that have a role in the regulation of protein-coding gene expression at post-transcription levels –30. MiRNMiRNA are also found secreted out of the cells as cell-free miRNA, packaged in exosomes, micro-vesicles or RNA-binding-proteins, that provide protection from RNases and serve as a new mode in cell communication . Cell-frThe miR-141 is a member of the miR-200 family, which is a family of miRNA that maintains epithelial cell integrity by inhibiting epithelial to mesenchymal transition (EMT). Several studies have reported the upregulation of miR-141 in cancers, such as colorectal cancer , prostatSince there are different results in the miR-141 expression among studies, we aimed to evaluate the expression of miR-141 in the blood plasma of patients with EOC and healthy subjects. The results could be helpful to evaluate miR-141 as a potential novel screening biomarker for EOC and other ovarian malignancies.This observational, analytical study used a cross-sectional design. All eligible patients with EOC diagnosed clinically, and confirmed pathologically, irrespective of age were included in this study. If patients had one of the following criteria, they were excluded from the study: i) patients with other organ cancers; ii) history of chemotherapy or radiotherapy; iii) pregnancy; iv) diabetes mellitus; and/or v) impaired renal function/diagnosed with renal disease.The sample size calculation was obtained from the following formula :r = Sample size ratio between EOC group and healthy control group is 1SD = Standard deviation of miR-141 expression in the blood plasma of cancer patient. Based on the previous study , standard = Expected mean difference of miR-141 expression between cancer and healthy control. Based on the previous study  from September 2015 to May 2016. Blood plasma samples obtained from 25 healthy women were used as the control group.Informed consent was obtained from each patient before the blood was collected. Five millilitres of blood samples were withdrawn from a peripheral venous puncture. The blood was collected using an ethylenediaminetetraacetic acid (EDTA) tube. Immediately after sample collection, the blood samples were centrifuged at 1,500 rpm for 10 min at 4 °C. Plasma was transferred into 1.5 mL RNase-free tubes using a tip filter and then the plasma was stored at −80 °C until further use.Total RNA extraction from 200 μL of blood plasma was performed according to the manufacturer’s instructions of miRCURY RNA Isolation Kit-Biofluid . Then, the concentration and purity were determined using a nanodrop spectrophotometer. The RNA was stored in a refrigerator −80 °C for reuse.A total of 4 μL of RNA isolated from plasma was reverse transcribed into complementary DNA (cDNA) using the universal cDNA synthesis kit II . The reverse transcription reaction was done by mixing the RNA, 5× reaction buffer, nuclease-free water, enzyme mix and spike in (sp6) for a total volume of 20 μL. Then, it was incubated at 42 °C for 60 min, with reverse transcriptase inactivation at 95 °C for 5 min, and cooling down at 4 °C. The cDNA was stored in a refrigerator at −20 °C.The real-time quantitative polymerase chain reaction (qRT-PCR) assay was accomplished using ExiLent SYBR Green master mix, 2.5 mL  and LNA miRNA PCR Primer Set (Exiqon) on Bio-Rad CFX96 Real-time Thermal Cycler System .The primer sequence for miR-141 was  . The priThe cDNA was diluted with RNase-free water at a ratio of 1:80 (1 μL cDNA with 80 μL RNase-free water). After that, 4 μL cDNA was then added with 5 μL SYBR Green master mix and 1 μL miRNA specific primer for a total volume 10 μL. The qRT-PCR was performed with the following conditions: early denaturation at 95 °C for 10 min, followed by 40 cycles 95 °C for 10 s, 60 °C for 1 min ramp-rate 1.6 °C/s optical read and analysed melting curve. The cycle of threshold (Ct) values were normalised using miR-16 as the reference gene. To assess the contamination, the no template control (NTC) was included at each qRT-PCR reaction.−ΔΔCt . The differences in expression level of miR-141 between patients with EOC and healthy controls were calculated using Mann-Whitney test, because the distribution of ΔCT miR-141 value was abnormal . The differences of expression level of miR-141 between early stage and advanced stage EOC were calculated using Mann-Whitney test, because the distribution of ΔCT miR-141 value was abnormal based on Kolmogorov-Smirnov test calculation (P < 0.05). The differences in expression level of miR-141 between Type I EOC and Type II EOC were calculated using Mann-Whitney test, because the distribution of ΔCT miR-141 value was abnormal . The P-value of less than 0.05 was considered statistically significant.Statistical analyses were performed using SPSS 23 . All numeric data were tested using Kolmogorov-Smirnov to assess the data distribution. We reported the data as mean and SD for normally distributed data, while the non-normally distributed data were reported as median and interquartile range (IQR). The age difference between patients with EOC and healthy controls was calculated using independent sample From September 2015 to May 2016, 30 female patients with histologically proven EOC were consecutively recruited for this study. A total of 25 healthy females was also recruited as a control group. Patients’ characteristics are presented in t-test, P = 0.533). In this study, the clinical stage of EOC was determined based on the FIGO staging system. Based on the clinical stage, 15 patients (50%) were diagnosed at an advanced stage, with 12 patients (40%) diagnosed with FIGO stage III and 3 patients (10%) diagnosed with FIGO stage IV. Based on histological types, serous and mucinous EOC were the most common types diagnosed in this study. Results showed 43.3% of patients were diagnosed with serous EOC, 36.7% of patients diagnosed with mucinous EOC, 6.7% of patients diagnosed with endometrioid EOC and 13.3% of patients diagnosed with clear cell EOC. All of the EOC patients in this study have a CA-125 level higher than the normal value (> 35 U/mL).The mean age was 47.73 years (SD = 10.29 years) in the EOC group and 44.48 years (SD = 16.14 years) in the healthy control group. There was no significant difference in age between the EOC and control group (P < 0.001). The relative expression of miR-141 in the blood plasma of EOC patients was upregulated 8.41 fold compared to the control group.The median ΔCt miR-141 was 9.37 (IQR = 2.53) in the EOC group and 11.64 (IQR = 1.49) in the healthy control group . The higP < 0.001). This result demonstrated that the expression of miR-141 was elevated significantly in the advanced stage compared to the early stage. The relative expression of plasma miR-141 in advanced stage EOC patients was upregulated 4.2 fold compared to early stage EOC patients.To determine the essential role of plasma miR-141 in the progression of EOC, we examined plasma miR-141 expression based on the clinical stage. For this purpose, we classified the clinical stage into two groups: early-stage (FIGO stages I and II) and advanced-stage (FIGO stages III and IV). As indicated in P-values of miR-141 between Type I and Type II EOC are presented in P = 0.592). This result suggests that the relative expression of plasma miR-141 is not significantly different between Type I and Type II EOC.Recent morphologic, immuno-histochemistry and molecular genetics studies suggest that there are two types of EOC: Type I and Type II . The hisOvarian cancer is ranked as the eighth major contributor to death in women due to cancer , which iBased on the histological subtype of EOC, serous is the most common type found, followed by mucinous. The serous subtype of EOC is the most common histological type in the world, especially in Western countries . Data obMinimal invasive biomarkers with high sensitivity and specificity for women with EOC are urgently required. In this study, we found that the relative expression of plasma miR-141 was upregulated significantly in patients with EOC compared to healthy subjects. Our results of the circulating miR-141 expression levels are consistent with a previous study, which confirmed that the miR-141 was highly-overexpressed in patients with EOC . Other pOur results suggested that miR-141 acts as onco-miR in EOC. Some studies have tried to elucidate the underlying mechanism of the miR-141 overexpression induced cancer phenotype. An epigenetic mechanism such as DNA methylation may cause deregulation of miRNA expression in cancer . The DNABromodomain containing 7 (BRD7) is a transcription factor that acts as a tumour suppressor protein in various cancers, including in ovarian cancer , 65. OveSingle miRNA can regulate multiple protein-coding genes, making miRNA exceptionally important regulators in a variety of cellular phenotypes . A recenIn this study, we found that miR-141 expression was elevated in advanced stage EOC. A study by Mak et al.  found thEffective screening strategy for EOC must not only have a high sensitivity for early-stage disease > 75%) but must also have a very high specificity (99.6%) to achieve an acceptable positive predictive value (PPV). Currently, CA-125 is widely used as a biomarker in clinical settings both for detection and disease monitoring in EOC. Although CA-125 is upregulated in the serum of most women with ovarian cancer, around 50% of stage I ovarian cancer cases have serum CA-125 level below the conventional cut-off of 35 U/mL . Besides% but musIn conclusion, this study demonstrated that circulating miR-141 levels were upregulated in patients with EOC compared with the expression levels in healthy subjects. Furthermore, miR-141 expression levels were also upregulated in advanced stage compared to early-stage disease. Our results indicated that circulating miR-141 is a potential minimally invasive biomarker for EOC detection and prognostication.


We find that the incorporation of Cr causes the strong hybridization between Li-2s, P-2p, and Cr-3d orbitals, resulting in a spin-polarized impurity band and forming stronger Cr-P polar covalent bonds. Li(Mg0.875Cr0.125)P becomes half-metallic ferromagnetism. The properties of the doped systems can be regulated by Li off-stoichiometry. When Li is deficient, the narrower impurity band and stronger p-d orbital hybridization enhance the half-metallicity. While the half-metallicity disappears, the band gap becomes wider, and the conductivity decreases for Li excess system, but its magnetic moments increase. Comparing optical properties show that the imaginary part of dielectric and complex refractive index function and optical absorption spectrum all have a new peak in the low energy region after Cr doping, and the new peaks are significantly enhanced when Li is deficient. The absorption range of low frequency electromagnetic wave is enlarged, and the energy loss functions show obvious red-shift effect for the doped systems. The results indicate that the properties of LiP can be controlled by Cr doping and Li off-stoichiometry independently, which will benefit potential spintronics applications.The electronic structures, magnetic and optical properties of Li Diluted magnetic semiconductors (DMS) combine the charge freedom and spin freedom of the electrons in the same matrix and have both advantages of semiconductor and magnetic materials. Although a lot of studies have been carried out for the traditional DMS As has higher Curie temperature (Tc) than As. Following this, Wang et al. (1.1(Zn1−xCrx)As, resulting in a ferromagnetic ordering below TC ~218 K, nearer to the room temperature. However, one shortcoming of Mn-doped and Cr-doped LiZnAs is the using of the toxic element Arsenic. The transition metal doped LiZnP P  are calculated by using the first principles method based on density functional theory. We find that the Cr doped systems exhibit half-metallic ferromagnetism. The electrical, magnetic and optical properties of LiP can be controlled by Cr doping and Li off-stoichiometry independently, which will benefit potential spintronics applications.In this work, the electronic structures, magnetic and optical properties of LiFLi1, VLi2, VLi3, and I Li1, I Li2, I Li3) have been designated in LiMgP is an antifluorite structure  code based on the density functional theory (DFT) method P, the geometric structure of the original LiMgP supercell is firstly optimized by local Density Approximation (LDA) and General Gradient Approximation (GGA), respectively. The obtained lattice constant, band gaps and total energies are listed in y1±(Mgx1−Crx)P are shown in The spin polarized band structures of Li0.875Cr0.125)P is 2.544 eV (0.875Cr0.125)P system become a half-metallic material with 100% spin-polarized ratio of conduction electron. The spin-flip band gap is 0.767 eV, which are much larger than many other 3d-transition-metal-element-based materials P is also an indirect band gap semiconductor with the band gap of 2.531 eV (as show in 0.875(Mg0.875Cr0.125)P is 0.854eV, and the half-metallicity is enhanced obviously when Li is deficient. Li excess system is also an indirect band gap semiconductor with the band gap of 2.560 eV. However, neither of the majority-spin and minority-spin impurity bands crosses the Fermi level. Therefore, Li1.125(Mg0.875Cr0.125)P change back to semiconducting nature again.Liy1±(Mgx1−Crx)P are shown in t2g and eg energy levels of Cr-3d state are separated from each other, and the t2g energy level is pushed toward the Fermi level, induce Li(Mg0.875Cr0.125)P to become half-metallic ferromagnetism. When Li is deficient, the electrons of P-3p and Cr-3d states have the stronger p-d orbitals hybridization P increases. When Li is excess, only a weak hybridization of p-d orbitals appears near the Fermi energy, and the t2g levels is completely occupied by electrons. The half-metallicity disappears.The calculated total and partial density of states (DOS) for Lidization . The t2gB, 2.98 μB, and 4.90 μB for the Cr doped, Li vacancy, and l Li excess system, respectively. The result indicates that the impurity band width and the net magnetic moments increase with the increasing of Li concentration.Moreover, the net magnetic moment is also calculated by integrating the occupied states below the Fermi energy and shown in y1±(Mgx1−Crx)P. For pure LiMgP P, the imaginary part of dielectric functions, complex refractive index functions, optical absorption spectra and energy loss spectra are also calculated and shown in E = 4.27 eV, corresponding to the direct transition of P-3p in valence bands to Li-2s, Mg-3s, and Mg-2p states in conduction bands. It is worth noting that a new peak occurs in the low energy region and the peak at E = 4.27 eV reduces after Cr doping. Moreover, we also find that the new peak is significantly enhanced when Li is deficient, but when Li is excess the new peak disappears, and the main peak moves slightly toward the low energy region. The refractive index for pure LiMgP is 2.571 (E < 1.82 eV) and higher energy (E > 11.54 eV) region, the imaginary part of the complex refractive index function is zero, and the real part is a constant, indicating that the absorption of LiMgP is limit to a certain frequency range. Similarly to the dielectric functions, there are also new peaks in the low energy region of complex refractive index functions after Cr doping, implying that the absorption range of low frequency electromagnetic wave is enlarged for the doped systems. It can be seen in 0.875Cr0.125)P, Li0.875(Mg0.875Cr0.125)P, and Li1.125(Mg0.875Cr0.125)P are about 63%, 29% and 138% of that for LiMgP, respectively.To further investigate the effects of Cr doping and Li off-stoichiometry on properties of Liis 2.571 . In the y1±(Mgx1−Crx)P  are calculated by using the first principles method based on density functional theory. We find that the Cr doped systems exhibit the half-metallic ferromagnetism. Due to the Cr doping, sp-d orbitals hybridization leads to spin-polarized impurity bands and form stronger Cr-P polar covalent bonds. Moreover, we also find that the properties of the doped systems can be regulated by Li off-stoichiometry. The impurity band width and the net magnetic moment increase with the increasing of Li concentration, but the half-metallicity and the conductivity decrease. When Li is excess, the p-d orbitals hybridization obviously becomes weaker and the half-metallicity disappears. Comparing optical properties shows that the imaginary part of dielectric, the complex refractive index function and the optical absorption spectrum all have a new peak in the low energy region after Cr doping, and the new peaks are significantly enhanced when Li is deficient. The absorption range of low frequency electromagnetic wave is enlarged, and the energy loss functions show obvious red-shift effect for the doped systems. The results indicate that the properties of LiP can be controlled by Cr doping and Li off-stoichiometry independently, which will benefit potential spintronics applications.The electronic structures, magnetic and optical properties of LiThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A highly porous nonwoven thermoplastic polyurethane (TPU)/Polypropylene (PP) triboelectric nanogenerator (N-TENG) was developed. To fabricate the triboelectric layers, the TPU nanofiber was directly electrospun onto the nonwoven PP at different basis weights . The surface morphologies and porosities of the nonwoven PP and TPU nanofiber mats were characterized by field-emission scanning electron microscopy and porosimetry. The triboelectric performance of the nonwoven TPU/PP based TENG was found to improve with an increase in the basis weight of nonwoven PP. The maximum output voltage and current of the TPU/PP N-TENG with 50% PP basis weight reached 110.18 ± 6.06 V and 7.28 ± 0.67 µA, respectively, due to high air volume of nonwoven without spacers. In order to demonstrate its practical application as a generator, a TPU/PP N-TENG-attached insole for footwear was fabricated. The N-TENG was used as a power source to turn on 57 light-emitting diodes through human-walking, without any charging system. Thus, owing to its excellent energy-conversion performance, simple fabrication process, and low cost, the breathable and wearable nonwoven fiber-based TENG is suitable for large-scale production, to be used in wearable devices.  Triboelectric nanogenerators (TENG) have attracted increased attention as effective self-powered devices for converting the ambient wasted mechanical energy into useful electric energy because of their high electrical output performance, high efficiency of energy conversion, simple structure, lightweight, and ease of fabrication ,7,8,9,10Fiber-based wearable TENG are one of the most challenging devices to develop for wearable electronics application. Moving away from traditional devices which use rechargeable batteries to a system of self-powering requires extensive design iterations. However, a fiber-based TENG has a distinct advantage: It has the inherent structural characteristics of fibers such as wearability, stretchability, breathability, and flexibility. Therefore, it can be a highly attractive platform for energy-harvesting ,17,18,19Melt blowing is a common method for producing nonwoven fabrics with a highly porous structure and high specific area. This process has the advantage of high production rate, low cost, and eco-friendliness (it is solvent-free), and it uses thermoplastics such as polypropylene (PP) , polyeth2) were manufactured by a melt-blowing process. Then, an electrospun thermoplastic polyurethane (TPU) nanofiber mat was directly fabricated on the nonwoven PP without any spacer; the porous structure served as a spacer. The morphological and structural characteristics of the nano- and microfiber-based nonwoven TPU/PP layers were investigated by field-emission scanning electron microscopy (FE-SEM), 3D digital microscope, and porosimetry. The triboelectric performance of the TPU/PP N-TENG fabricated with different basis weights of PP was also evaluated.In this study, we developed a highly porous nonwoven fiber-based nanogenerator without a spacer for energy harvesting. Nonwoven PPs with various basis weights  purchased from BASF Corp., (Germany) was used to fabricate the nanofiber web. A mixture of N,N-dimethylformamide (DMF) and tetrahydrofuran (THF) purchased from Thermo Fisher Scientific (USA) was used as the solvent for preparing the TPU solution.PP  with a density of 0.9 g/cm2.In order to fabricate the microfiber-based nonwoven PP fabric, the melt-blowing process was performed with a pilot-scale melt-blow line . PP resin was melted at 250 ℃ in the extruder, and the polymer melt was extruded through a spinneret, with an air gap of 0.4 mm. The nozzle to collector distance was 300 mm. The blowing-air temperature to obtain the nonwoven fabric was set to 260 ℃. The melt-blown nonwoven PP fabrics were fabricated with different basis weights of 15, 30, and 50 g/mTo form the TPU nanofiber mat on the nonwoven PP fabric, TPU was dissolved in a mixture of DMF and THF (7:3), to obtain a 12 wt% solution. The dissolved solution was electrospun onto the obtained nonwoven PP fabric, without any spacer, through the metal nozzle (25G) connected to a high-voltage generator . The applied voltage and feed rate were 18 kV and 0.3 mL/h, respectively. The distance from the metal nozzle to collector was 15 cm. After that, the so-obtained nano- and microfiber nonwoven triboelectric layer was dried at 30 ℃, under vacuum, for 24 h.The triboelectric layer consisted of the nonwoven PP fabric (161–475 μm thick) and TPU nanofiber mat (80 μm thick) as electron-acceptor and electron-donor materials, respectively. The two triboelectric layers were directly fabricated by the spinning process without further processing. The porous nonwoven PP/TPU triboelectric layers were cut into 50 mm × 50 mm pieces. One of the pieces was placed between two Ni-coated conductive fabric (45 mm × 65 mm × 0.11 mm) serving as electrodes, and the structure was fixed with conductive adhesives. The so-fabricated TPU/PP N-TENG was examined in the vertical contact mode.oc) and short-circuit current (Isc) of the TENGs with various basis weights of the nonwoven fabric were tested with a constant force of 5 N, at a frequency of 8 Hz. The porous nonwoven TPU/PP50 TENG was examined under different external forces and frequencies. Finally, the N-TENG-based insole was connected to 57 light-emitting diodes (LEDs) and tested under different human motions, such as walking and running.The surface and cross-sectional morphologies of the nonwoven PP and TPU nanofiber mat were examined by FE-SEM  at an acceleration voltage of 10 kV, after sputter-coating with osmium. The surface and internal topology were also observed by using a 3D digital microscope . In addition, the data for the curved surface diagram were extracted from the cross-sectional topology image. The pore size distribution and porosity of the porous nonwoven PP and TPU nanofiber mat were characterized by a capillary flow porometry , according to ASTM F316-03, and mercury porosimetry , respectively. The permeability of the porous nonwoven PP and TPU nanofiber mat was measured by an air permeability tester . The electrical output performance of the porous nonwoven fiber-based TPU/PP N-TENGs was assessed with a digital oscilloscope  and a source meter . The open-circuit voltage  and short-circuit current (Isc) of the TPU/PP50 N-TENGs under different external forces at a fixed frequency at 8 Hz. The results indicate that the Voc and Isc increased from 1 to 5.8 V and from 0.15 to 1.1 μA, respectively, as the external force increased from 0.1 to 5 N. It is clear that the output performance of the N-TENG improves with an increase in the external force. At a lower external force, the fibers of each triboelectric layer make contact over a relatively smaller area in comparison to that achieved under higher external force. As the force increases, a large deformation of N-TENG takes place, leading to increased contact area between the triboelectric layers. This explains why the contact area between the triboelectric layers increases when the applied force is increased, resulting in the improvement of the output performance. Since wearable devices with a nanogenerator are always exposed to physical energy with irregular and varying frequencies, it is essential to evaluate the frequency-dependence of the TENG device [oc and Isc of the TENG increase from 1 to 3.63 V and 0.17 to 1.1 μA, respectively, presenting a clearly increasing trend with the frequency in the range of 0.5 to 8 Hz. The fast deformation frequency allows electrons to flow rapidly through the external circuit to reach equilibrium in a short time [2 is reached at a load resistance of 10 MΩ (G device . Figure of 10 MΩ f.In general, a person usually walks more than 6000 steps per day. In addition, while walking, a person’s load is periodically applied to the foot at an average frequency of 1.8 Hz. Therefore, the walking-based generator is advantageous for generating electrical energy by friction. As shown in In summary, we developed highly porous nonwoven TPU/PP-based TENGs, which are flexible and breathable, for wearable devices. We investigated the influence of the basis weight of nonwoven PP on the triboelectric performance of TENGs. Structural characterization revealed that the micro-sized PP nonwoven has a relatively large pore size and high permeability in comparison to that of its counterpart, TPU nanofiber mat, and the porous structure and thickness of nonwoven PP were affected by the basis weight. Owing to the highly porous nonwoven structure with a high air volume, the interface layer between the two counter triboelectric layers were not fully in contact even when they were integrated. Thus, the TENG could harvest energy under different external forces and frequencies. Under walking and running motions, our TPU/PP50 N-TENG provided an output voltage and current of 110.18 ± 6.06 V and 7.28 ± 0.67 µA, respectively. Furthermore, the energy generated by our N-TENG-based insole could be used to directly light up 57 green LEDs sustainably. Thus, we fabricated a facile, breathable, and flexible generator fully based on fibers, which can be applied in wearable devices. In addition, the manufacturing method of the triboelectric layer of our N-TENG is low-cost and amenable to mass production. Therefore, this study can help develop large-area generators that can be applied in self-powered wearable clothes and footwear in the near future.


Staphylococcus aureus (S. aureus) infections affect SP-A expression. To closely reflect the situation in the lung, we used a human alveolus-on-a-chip model and a murine pneumonia model. Our results show that S. aureus can reduce extracellular levels of SP-A, most likely attributed to bacterial proteases. Mono-epithelial cell culture experiments reveal that the expression of SP-A is not directly affected by IAV or S. aureus. Yet, the mRNA expression of SP-A is strongly down-regulated by TNF-α, which is highly produced by professional phagocytes in response to bacterial infection. By using the human alveolus-on-a-chip model, we show that the down-regulation of SP-A is strongly dependent on macrophages. In a murine model of pneumonia, we can confirm that S. aureus decreases SP-A levels in vivo. These findings indicate that (I) complex interactions of epithelial and immune cells induce down-regulation of SP-A expression and (II) bacterial mono- and super-infections reduce SP-A expression in the lung, which might contribute to a severe outcome of bacterial pneumonia.Pneumonia is the leading cause of hospitalization worldwide. Besides viruses, bacterial co-infections dramatically exacerbate infection. In general, surfactant protein-A (SP-A) represents a first line of immune defense. In this study, we analyzed whether influenza A virus (IAV) and/or  Staphylococcus aureus (S. aureus), a facultative-pathogenic bacterium, is of major importance. S. aureus colonizes epithelial surfaces, but can also cause a broad spectrum of infections ranging from superficial skin infections to life-threatening diseases, such as bacterial pneumonia [Pneumonia is the most severe inflammatory disease of the lower respiratory tract and also the most common infectious disease worldwide. A wide variety of microorganisms can induce pneumonia, including viruses and bacteria. Among these, influenza A and B viruses  are primary causative agents . The higneumonia .The first line of defense during infections, apart from physical barriers, is regulated by an innate host immune response in which white blood cells and chemical components play a crucial role . GranuloS. aureus [S. aureus by neutrophils and macrophages and inhibits bacterial adherence to the epithelial layer, thus preventing the bacterial penetration into the host cells [Surfactant proteins interact with several pathogens, among others, with IAV  and S. a. aureus . During . aureus . Surfact. aureus . The agg. aureus . Furtherst cells . Several inflammatory cytokines alter SP-A expression. Among these, TNF-α is known as a strong suppressor of SP-A ,15,16,17S. aureus expresses a cysteine protease staphopain A  that cleaves SP-A and inactivates the antibacterial activity of the protein, promoting the colonization of the lung by S. aureus and other pathogens [Yet, bacteria can also directly affect pulmonary surfactant proteins , as S. aathogens . S. aureus or the combined infection of both pathogens has an impact on the SP-A expression in the lung, which could represent a mechanism to weaken the host defense system and worsen the outcome of a lung infection. Taken together, many parameters during an infection affect the SP-A expression. In this study, we aimed to elucidate whether IAV, For the single cell-culture, we use alveolar epithelial cells. The human adenocarcinoma cell line NCI-H441  was cultivated in RPMI-medium  and supplemented with 10% inactivated fetal bovine serum . The cell line was sub-cultured in the laboratory and tested for contamination with mycoplasma. NCI-H441 cells were grown up to 80% confluence before being sub-cultured and used until the 18th passage.6 monocytes/well were cultured in 24-well dishes in RPMI-medium supplemented with 10% autologous human serum, 10 ng/mL human granulocyte-macrophage colony-stimulation factor , 10 ng/mL macrophage colony-stimulation factor , and penicillin/streptomycin. The cells were incubated for 1 h at 37 °C and 5% CO2 and then washed twice with RPMI. Cells were cultured for 6 days. A medium exchange was performed every second day. RPMI-medium was supplemented with 10% autologous human serum, 10 ng/mL GM-CSF, to induce macrophage differentiation, and penicillin/streptomycin.Besides this, primary monocytes were isolated from whole peripheral blood of healthy donors. The blood was diluted in an equal volume with phosphate buffer saline  and then gently transferred to the surface of Biocoll separation solution . The monocytes were separated from blood cells by centrifugation without bakes. Cells were washed with PBS three times with centrifugation steps in-between. For cell culture experiments, 6 × 10Macrophage differentiation from monocytes was routinely checked by staining for macrophage cell type markers CD68, and CD163 in random-wise selected control samples. No significant donor-related variation of differentiation efficiency or cell numbers was observed. 5 pores/cm2. The capacity of the endothelial is 220 µL (including afferent and efferent channels), and the volume of the epithelial chamber is 120 µL . Peristaltic pumps were used for the perfusion of the biochip, as explained previously [For the establishment of the human alveolus-on-a-chip model, we used MOTiF biochips, manufactured and purchased by microfluid ChipShop GmbH . The chip consists of two chambers that are separated by an 11 µm thick polyethylene-terephthalate (PET) membrane with a pore diameter of 8 µm and pore density pf 2 × 10eviously .2 [For the cell cultivation, we use NCI-H441 cells, endothelial cells  and human monocyte-derived macrophages (hMdM) as described above cm2 .5 NCI-H441/cm2. Cells were cultured for 3 d until confluency with a daily media exchange. The NCI-H441 medium was supplemented with 1 µM dexamethasone . NCI-H441 cells  were seeded on the opposite side of the membrane  with a density of 2.7 × 105 macrophages/cm2  [HUVECs were kindly provided by Regine Heller  and were isolated from human umbilical cord veins as described previously and seeded into the biochip (vascular cavity) at a density of 2.7 × 10Germany)  for 48 hGermany) . 2. After freezing and a centrifugation step, the virus was stored at −80 °C.For the propagation of IV, we used Madin-Darby canine kidney (MDCK) cells. MDCKs were cultured in EMEM , supplemented with 10% fetal calve serum (FCS). For the virus propagation, the IV strain A/Puerto Rico/8 was passaged on MDCK in EMEM supplemented with 25 mM MgCl, 1.3% bicarbonate, and 0.333% trypsin. The infected cells were incubated at 37 °C and 5% COFor the murine model, the A/H1N1/pdm09 influenza virus variant HA-D222-mpJena/5258, expressing an aspartate in position 222 of the viral hemagglutinin, was passaged on MDCK. HA-D222-mpJena/5258 was obtained after three plaque-purification steps  from theS. aureus/USA300/wild type (WT), kindly provided by Lorena Tuchscherr [S. aureus USA300 was grown overnight in brain heart infusion (BHI)-medium at 37 °C with shaking. All bacteria were stored in the BHI medium at −80 °C. Colony-forming units (CFU) of S. aureus were determined weekly to calculate the number of infectious particles in the cryopreserved bacterial working solution and to exclude changes in the number of living bacteria by freezing.The bacterial infection was performed with a methicillin-resistant chscherr . The bacS. aureus load after the infection was performed by using the supernatants. For this, the supernatants were serial-diluted in PBS and plated on blood agar plates . After overnight incubation at 37 °C, colonies were counted to detect the number of bacteria.The determination of the 2, 0.9 mM CaCl2) and incubated for 30 min at 37 °C without centrifugation. For the specification of the pathogen concentration, we have used the multiplicity of infection (MOI). This information describes the ratio of pathogens to infected cells. For the viral infection of the NCI-H441 cells we use MOI 1. For the viral infection of the NCI-H441 mono-cell culture as well as the infection on the human alveolus-on-a-chip model, cells were washed with PBS once  . The IV 2. Afterwards, the cells were washed. To eliminate the non-internalized bacteria, cells were treated for 20 min with RPMI-medium containing lysostaphin at a concentration of 6 µg/mL for the NCI-H441 mono-cell culture and 20 µg/mL for the human alveolus-on-a-chip model. Subsequently, cells were washed with PBS and then incubated with RPMI for indicated time points at 37 °C with 5% CO2.The bacterial infection on NCI-H441 cells was performed with RPMI  with 1% human serum albumin (HSA) and 1mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The epithelial side of the human alveolus-on-a-chip model was infected with RPMI/BA  with bacteria (MOI 5). After the bacterial infection was performed, cells were incubated with bacteria for 90 min at 37 °C and 5% COS. aureus/USA300/WT (MOI 5).For the co-infection scenarios, both pathogens were added to the cells. For this, first, the infection with IV strain A/Puerto Rico/8 (MOI 1) was performed, followed by the bacterial infection with 2, 0.9 mM CaCl2) and incubated for 30 min at 37 °C without centrifugation until the indicated time points. For the single bacterial infection as well as the co-infection, we use S. aureus/USA300/WT (MOI 1).For the infection of the single cell-culture of hMdM, cells were isolated as described above and used after 6 days of cultivation. After a washing step with RPMI, the IV strain A/Puerto Rico/8 (MOI 1) was added to the cells in RPMI , a single infection with the virus, a single infection with bacteria, and a co-infection. All cells were treated with the same medium. This medium contained the according pathogen for the single- or co-infection or no pathogens for the mock-treated cells of the cell control.For the murine pneumonia model the mice were purchased from Janvier Labs . The animal application was proven by the local ethics committee of the Thuringian State Office for Consumer Protection . The mice were maintained according to institutional guidelines in individually ventilated cages, and food and water were given ad libitum. For the in vivo model, we use specific-pathogen-free (SPF) C57Bl/6 mice from Janvier Labs , providing us with an excellent health status for the animals. The animal facility of the Jena University Hospital also provides an excellent unit for performing animal experiments. Under S2 conditions, the animals are housed in separate units, which are monitored frequently.S. aureus USA300 or with both pathogens in a co-infection. Therefore, the mice were anesthetized by isoflurane inhalation and intranasally inoculated with HA-D222-mpJena/5258 (5 × 104 plaque-forming units (PFU)) in 20 μL NaCl or only 20 µl NaCl (mock-treated). The same method was used for S. aureus infection (1 × 107 CFUs) two days later. During the infection period, the state of the animal health was monitored up to two times per day by controlling the weight, temperature and the scoring of the general behavior condition. Four days after HA-D222-mpJena/5258-infection, animals were sacrificed, and samples of blood and lung were subjected to further analysis. The viral titer and the bacterial counts of infected mice were determined in the right inferior lung lobe. Eight-week-old female C57bl/6 mice were infected with the IV strain HA-D222-mpJena/5258, Moreover, the right superior lung lobe was used to determine the mRNA expression of TNF-α and SP-A by quantitative real-time PCR. For histopathology, the left lung lobe of each mouse was fixed in 10% formalin for at least 24 h, dehydrated in a graded series of alcohol and xylene, and mounted in paraffin. For each lung sample, about ten 5 µm thick sections were stained with hematoxylin and eosin (HE) to study the degree of cellular infiltration. Sections of each lung were examined microscopically (Zeiss microscope Axio Vert.A1), and representative photographs were obtained (Axio Cam ERc5S).−ΔΔCt method [For quantitative real-time PCR, cell infection was conducted as described above. Isolation of total RNA from macrophages, NCI-H441 and homogenized lungs was done by using the RNeasy Mini Kit  according to the manufacturer’s instructions. Cells of the biochip model were first homogenized using the QIAshredder homogenizers (Qiagen) and total RNA was isolated with RNeasy Micro Kit (Qiagen) following manufacturer’s instructions. Equal amounts of RNA were transcribed into cDNA using QuantiNova Reverse Transcription Kit (Qiagen) according to the manufacturer’s protocol. Gene expression of several genes was determined by qRT-PCR on a Thermo Scientific™ PikoReal™ Real-Time PCR System using QuantiTect SYBR Green (Qiagen) and gene-specific primers . Relativt method .To analyze the mRNA expression of SP-A, we have compared the data from the infection with the pathogens and the data of the stimulation with recombinant TNF-α. For this, we used 10 nM of TNF-α  added to RPMI. After 30 min of incubation on NCI-H441 cells, they were washed with PBS and incubated for 4 h. Afterward, the mRNA expression was performed as described above.For the detection of extracellular TNF-α we performed the infection of the cells with virus, bacteria and both pathogens as described above (compare with the chapter on in vitro infection) and determined the concentration of TNF-α within the supernatants.450 was determined by the microplate reader SPECTROstar Omega . The protein concentration was calculated by comparison to a standard curve. Supernatants were collected at 30 min and 4 h p.i. and transferred to an antibody-coated 96-well plate in duplicates  and subsequently analyzed according to the manufacturer’s instructions. For the identification of SP-A, we used NCI-H441-cells and performed the infection as previously described . Next, 4For the measurement of pro-inflammatory cytokines, a LEGENDplex™ Human Inflammation Panel (13-plex)  was used. Alveolar biochips were infected as previously described and 4 h p.i. supernatants were collected. 25 µL of each sample was transferred in duplicates into the 96-well filter plate and the LEGENDplex panel was performed following the manufacturer’s instructions. Samples were measured the same day on a flow cytometer , and the cytokine amount was calculated by comparison to a standard curve. For the immunofluorescence staining of the cells of the human alveolus-on-a-chip model, the membranes with the NCI-H441 cells were fixed with 4% paraformaldehyde at room temperature. For the staining of the endothelial sides, cells were fixed for 15 min with ice-cold methanol at −20 °C. Afterwards, intracellular staining of SP-A was performed. For this, membranes were incubated overnight at 4 °C in PBS containing 0.1% Saponin, 3% donkey serum and anti-SP-A antibodies . Thereafter, fluorescent labeled secondary antibodies  were used and incubated for 30 min. The stained samples were embedded into a mounting medium . Imaging was performed with an Axio Observer Z1 fluorescence microscope with ApoTome.2 extension , and images were analyzed with ImageJ2 software (Fiji distribution).2 and sputter coated with gold (thickness approx. 2 nm) using an SCD005 sputter coater  to avoid surface charging. Finally, the specimens were investigated with a field emission (FE) SEM LEO-1530 Gemini .The fixation of the cells was performed inside the human alveolus-on-a-chip model by using a fixative solution  for 90 min. Afterwards, a fresh cacodylate buffer was used for 30 min to wash out the fixative. The membranes were dehydrated in ascending ethanol concentrations  for 15 min each. The samples were critical point dried using liquid COFor the analysis, images were used as Z stacks in the Zeiss native image format “CZI”. After deconvolution with Huygens Software , the images were segmented in Imaris 9.2.1 . The complete process was published before .All experiments were performed in technical duplicate and at least three were independent experiments. After an examination of quality criteria, non-parametrical methods were used for analysis. Statistical significances were evaluated by one-way-ANOVA  followed by Mann–Whitney U test or by using one-way-ANOVA Tukey’s multiple comparison test. Statistical analysis was performed using SPSS Statistics 25 software  and Prism software .To determine the effect of the mono- and co-infections on the SP-A production we used single cell cultures of alveolar epithelial cells type II (NCI-H441 cells). To verify an efficient infection, viral titers A, as welS. aureus and after co-infection, SP-A mRNA-expression was not changed. By contrast, treatment with TNF-α, a well-known suppressor of SP-A, resulted in significantly decreased SP-A mRNA expression , phenol-soluble modulin (PSM) [After mono-infection with IAV or pression C. Next, pression D. This fping Kit . Here, win (PSM) , and supin (PSM) .During viral and/or bacterial infection, the cytokine profile, including the expression of TNF-α, of infected cells is altered depending on the host cell type and on the pathogens. In an epithelial mono-cell culture (NCI-H441 cells) bacterial infection caused a much lower increase in TNF-α mRNA-expression compared to the infection of immune cells (hMdM) . Here, wMacrophage differentiation from monocytes was proven by performing single-cell experiments with hMdM. Here we can demonstrate that macrophages react on their mRNA expression level after challenging with pathogens. All cells infected with virus, bacteria or both, had a significantly higher level of CD80 (mRNA) than the mock-treated cells . By contS. aureus infection was much higher in macrophages than in epithelial cells, whereas IAV-infection did not significantly increase the TNF-α expression in either cell types , endothelial cells (HUVECs) and macrophages (hMdM) were co-cultured under perfusion of the endothelial side and infected via the epithelial side with the pathogens as indicated. The successful viral infection was proven by the budding process of IAV, as visible in the SEM-pictures A. Furtheip model A. In thi release B. S. aureus or co-infection  or independent (IAV infection) on SP-A expression.Our results show a high viral load (PFU/mL) 4 days p.i. A. SimilaSecondary bacterial pneumonia is one of the main reasons for increased morbidity and death rates after IAV infections. Apart from the pathogen load caused by the super-infection, the dysregulated immune response is also involved in the severe outcome of concomitant bacterial and viral infections. The pulmonary surfactant plays an important role as the first line of defense. The aim of our study was to elucidate whether a viral/bacterial co-infection has an impact on SP-A expression, the major surfactant protein.SP-A fulfils many functions in the innate immune host defense. It regulates the inflammatory chemotaxis via the collagen-like region which induces an increased macrophage migration. Furthermore, SP-A is able to enhance phagocytosis as well as killing by macrophages and neutrophils via agglutination and opsonization of pathogens .The deficiency of surfactant proteins can lead to various diseases. First of all, in premature infants, the insufficient surfactant production causes neonatal respiratory distress syndrome (RDS) . A decreFurthermore, the adult acute respiratory distress syndrome (ARDS) is also associated with a decreased amount of SP-A and surfactant phospholipids . In agreThese examples demonstrate the great importance of a sufficient surfactant level in the lung and also the possibility to improve the lung function by external surfactant administration during a state of deficiency.S. aureus disposes of extracellular proteases with a wide variety of virulence-associated functions. Here, metallo-, cysteine and serine protease lead to tissue degradation and interference of the host immune response. The protease ssPA was described as the most efficient in degrading SP-A, even in a nanomolar concentration [S. aureus proteases impairs the bacterial clearance and results in enhanced adherence and invasion of S. aureus to epithelial cells [ntration . The diral cells . Additioal cells . HoweverS. aureus infection hampers the function of SP-A. It is well known that S. aureus infections lead to a high release of TNF-α, in particular originating from immune cells, such as macrophages. Yet, TNF-α expression was not increased upon IAV infection in immune cells within the present experimental setting. The regulation of SP-A is dependent on cytokines, in particular on TNF-α. TNF-α is capable of altering the mRNA level of SP-A via the phosphorylation of p38 mitogen-activated protein kinases ,47. As aAlthough SP-A is mainly produced by alveolar-epithelial type II cells, other cell types, such as endothelial and immune cells, are in close contact with each other within the alveolus. The different cell types interact via cytokines and influence each other, which cannot be reproduced in vitro in a mono-cell culture system. Consequently, the response to infection in the lung is highly complex and requires appropriate model systems where different cell types interact with each other. Frequently used models to study infections are mice. Here, the infection can be analyzed in the complete organ, including damages in the alveolar structures. Yet, the mice models have some disadvantages. (i) The cells are of murine origin and in particular immune functions can largely differ from human cells. For example, the action of defined bacterial toxins  can be very species-specific and largely restricted to human cells ,49,50. (S. aureus reduces SP-A protein levels, as S. aureus expresses the cysteine protease sspA which is able to cleave SP-A [S. aureus directly affects the mRNA expression of SP-A in NCI-H441 mono-cell culture, indicating that bacterial and viral pathogens do not affect the SP-A expression within the epithelial cells. Furthermore, our results demonstrate that TNF-α is a strong inhibitor of SP-A mRNA-expression. Bacterial mono- or co-infection strongly induce the expression of TNF-α, in particular in hMdM cells. By contrast, IAV infection has only a minor effect on TNF-α synthesis, which can be explained by the fact that IAV is inhibited by TNF-α and therefore TNF-α is disadvantageous for the survival of the virus particles [By combining the different model systems in our present study, we demonstrate that ave SP-A . Howeverarticles . The intS. aureus or both pathogens in the lung that caused inflammation in all cases. In line with our in vitro results, we could clearly demonstrate that the SP-A mRNA expression was down-regulated, particularly in the lung tissue of the bacterial-infected mice. Additionally, we have performed in vivo studies. Mice were infected with IAV, S. aureus leading to the cleavage of extracellular SP-A, there is an indirect effect based on infection via the cytokine production, notably by TNF-α. During infection, macrophages are one of the main sources for elevated TNF-α levels that have already been described to lower mRNA levels of SP-A. Our results show that beside the direct effect of Therefore, we concluded that an interaction of macrophages and alveolar epithelial cells decrease SP-A mRNA levels. These data demonstrate that the analysis of infection processes requires the use of sophisticated model systems that are based on various human cells and mimic the organ system.Pneumonia is the leading cause of morbidity and mortality worldwide and viral/bacterial co-infection worsens the severity of the disease. The innate immune response is a crucial part of the defense against these pathogens. Surfactant proteins are a part of this mechanism and have a major effect on the alveolar homeostasis.S. aureus infection on the SP-A expression in vitro and in vivo. We can show that complex interactions of epithelial cells and immune cells result in the down-regulation of SP-A mRNA expression. Furthermore, bacterial mono- and super-infections reduce the SP-A expression in a murine model which might contribute to the severe outcome of bacterial pneumonia.Our study investigated the impact of IV and/or